The hypothesis to be tested is that lysophosphatidic acid (LPA) inhibits secretory diarrhea through CFTR-dependent protein interactions. The long-term objectives of this laboratory as related to this grant are (i) to gain a better understanding of the dynamic protein-protein interactions that regulate LPA-dependent inhibition of CFTR and (ii) to understand the relevance of these interactions in secretory diarrhea. The specific aims of the grant are (AIM 1) to test the hypothesis that LPA inhibits cholera toxin-induced and CFTR-dependent secretory diarrhea in mice and (AIM 2) to test the hypothesis that a macromolecular complex consisting of LPA2, CFTR, and NHERF2 is required for the LPA-elicited inhibition of CFTR-dependent Cl-transport. To advance the research mission of the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), the proposed research will yield important basic science information essential to understanding, treating, and preventing digestive diseases such as secretory diarrhea. In Aim 1, we will test the hypothesis that LPA inhibits cholera toxin-induced and CFTR-dependent secretory diarrhea in mice. In subaim 1a, we will test whether LPA inhibits CFTR function in cultured gut epithelial cells and in excised mouse intestinal tissue. In subaim 1 b, we will test whether LPA inhibits cholera toxin-induced CFTR-dependent secretory diarrhea. In subaim 1c, we will test whether LPA does not inhibit CFTR function in LPA2 receptor knockout mice. In Aim 2, we will test the hypothesis that a macromolecular complex consisting of LPA2, CFTR, and NHERF2 is required for the LPA-elicited inhibition of CFTR-dependent Cl-transport. In subaim 2a, we will determine if LPA2, CFTR, and NHERF2 are assembled in a macromolecular complex in vitro. In subaim 2b, we will cross-link the components of the preexisting macromolecular complex (LPA2, CFTR, and NHERF2) in cultured epithelia and in mouse intestinal epithelial cells. In subaim 2c, we will test whether LPA inhibits the CFTR Cl-transporter due to a physical interaction between LPA2, and CFTR (mediated by NHERF2). At present, the molecular mechanisms responsible for LPA-mediated inhibition of secretory diarrhea are unclear. This project is a critical step in understanding the molecular mechanisms underlying the beneficial effects of LPA, thereby making possible improved treatments in the prevention of secretory diarrhea.